Post-translational modifications of cellular proteins by the small ubiquitin-like modifier (SUMO) family of proteins are important epigenetic mechanisms for regulating various cellular functions. At least three members of the SUMO family (SUMO-1, -2, and -3) are ubiquitin-like proteins that can conjugate to other cellular proteins by a biochemical mechanism similar to ubiquitination (Hay 2005; Sarge 2009; Yeh 2009).
SUMOylation requires multiple steps that are catalyzed by three types of SUMOylation enzymes: activating enzyme E1 (made up of two subunits, SAE1 and SAE2/Uba2), conjugating enzyme E2 (Ubc9), and one of several E3 ligases. This pathway is illustrated for SUMO 1 in FIG. 1. Briefly, SUMO is activated by the E1 enzyme through ATP hydrolysis and forms a thioester conjugate with E1. SUMO is then transferred to E2, forming a thioester conjugate with E2. Finally, SUMO is transferred to target proteins, a step usually stimulated by an E3 ligase. SUMO modification adds a new docking site to target proteins, and thus enables new protein-protein interactions through the SUMO-interacting motif (SIM) in receptor proteins (Song 2004; Song 2005). The E1 and E2 enzymes do not discriminate among the different SUMO paralogues (Tatham 2003).
SUMOylation is reversible by a process known as deSUMOlyation. The removal of SUMO proteins from modified target proteins is accomplished by deSUMOylation enzymes such as isopeptidase and SUMO/sentrin-specific protease (SENP).
Aberrations in post-translational modification of cellular proteins by the small ubiquitin-like modifier (SUMO) family of proteins are associated with the pathogenesis of life-threatening diseases, such as cancer (Luo 2009; Kim 2006; Mo 2005), neurodegenerative disorders (Steffan 2004; Subramaniam 2009), and viral infection (Jaber 2009; Ulrich 2009; Kim 2010). Viral infection often involves hijacking the host post-translational modifications, providing viruses with a fast means for becoming established in host cells before the immune system can respond.
SUMOylation and deSUMOylation enzymes regulate dynamic SUMO modifications in controlling cellular functions. One of the predominant functions of SUMO-mediated modifications is in DNA damage response, such as damage caused by chemo- and radiation therapy (CRT), which kills cancer cells by inducing genotoxic stress (Galanty 2009; Morris 2009; Ouyang 2009; Prudden 2009; Li 2010). DNA double-strand breaks (DSBs) are the most dangerous form of DNA damage, and lead to cell death if left unrepaired (FIG. 2) (Darzynkiewicz 2009). Upon DSB formation, the histone protein H2AX is phosphorylated, resulting in recruitment of several DNA damage signaling proteins to the damage sites, including p53-binding protein 1 (53BP1) and ATM (van Attikum 2009). SUMOylation is required for multiple steps in DNA repair pathways, including recruitment of signaling and repair proteins to damage sites and enablement of repair protein function. For example, recruitment of 53BP1 to DNA damage sites is dependent on its SUMOylation (Galanty 2009). 53BP1 mediates the signaling process to stop cell cycle progression and DNA replication and allows time for repair, in which p53 plays an important role. p53 is also involved in apoptosis if DNA damage is not repaired. SUMOylation also plays a role in regulating p53 transactivating activity (Stehmeier 2009) and trafficking (Carter 2007).
SUMOylation also directly regulates repair of various types of DNA damage. Recent studies have shown that SUMOylation is required for both major DSB repair pathways: homologous recombination (HR), in which a homologous sequence acts as a repair template, and non-homologous end joining (NHEJ), in which DSB ends are ligated together (Jeggo 2009). Proteins involved in HR include the well-known breast cancer-related genes BRCA1 and BRCA2, as well as other proteins with DNA binding and helicase activities (Jeggo 2009). Proteins that carry out NHEJ include Ku70, Ku80, DNA-PKcs, XRCC4, XLF, and Artemis (Jeggo 2009). Many proteins in the DSB repair pathways are substrates of SUMOylation (FIG. 2) (Doksani 2009; Morris 2009; Bartek 2010; Li 2010). SUMOylation is also important for response to single-stranded DNA damage (Pfander 2005) and nucleotide base excision repair (Steinacher 2005; Mohan 2007) by modifying repair enzymes to regulate their activity and life spans. These findings suggest that inhibition of SUMO-dependent processes can inhibit repair of a wide range of DNA damage in cancer cells, thereby sensitizing tumor cells to genotoxic stress induced by CRT.
SUMOylation is required for DNA repair, as evidenced by the observation that cells defective in SUMOylation are sensitive to DNA damage reagents (al-Khodairy et al. 1995; Shayeghi et al. 1997). Recently, two independent studies have identified the yeast protein, Mms21, as the SUMO E3 ligase required for repair of both DNA alkylation damage and double-strand breaks (Andrews et al. 2005; Zhao & Blobel 2005). Elimination of Mms21's SUMO E3 activity leads to DNA damage sensitivity. However, the SUMOylation targets in the DNA damage response are not yet well established, nor is SUMOylation's involvement in DNA repair or other cellular functions. Recent studies have shown that a SUMO-targeted ubiquitin ligase (STUBL) is important in DNA damage response, and the ligase specifically recognizes poly-SUMO-2/3 chains to ubiquitinate poly-SUMO modified proteins for degradation (Burgess et al. 2007; Ii et al. 2007; Prudden et al. 2007; Nagai et al. 2008; Cook et al. 2009; Sun et al. 2007).
The enzymes catalyzing SUMO-modification (E1, E2, E3) are present in higher levels in cancer tissues versus normal tissues and in metastasized tumors versus normal cells, and play an important role in cancer proliferation and metastasis. Recent studies suggest that E1 presents an ideal target for the development of cancer therapeutics with specific genetic backgrounds. For example, a genome-wide siRNA screen identified the genes encoding the SUMO E1 subunits SAE1 and SAE2 among those genes with the strongest synthetic lethal interactions with KRas (Luo 2009).
DeSUMOylation enzymes are also thought to be important in cancer. Increased levels of a deSUMOylation isopeptidase (Senp1) have been observed in prostate cancer, and suppression of Senp1 level by siRNA has been shown to suppress prostate cancer and angiogenesis. Hypoxia also induces high levels of SUMO-1. SUMO-mediated protein-protein interactions appear to be involved in most SUMO-dependent processes.
Given the role of SUMOylation in cancer and other disease states such as viral infection, there is a need in the art for novel SUMOylation enzyme inhibitors. Such inhibitors would be useful both as therapeutics and as research tools for studying the role of SUMOylation in cellular regulation.